Vaccine preparation from single antigen or biomarker has limited immunogenicity to provide desired/required protective immunity. To achieve broad spectrum protection a combination/repertoire of antigens is required. Such a combination is possible by the use of whole cells as they comprises of different immunogenic markers and antigens.
Cancer cells if used live as vaccine can cause cancerous growth and can imbibe disease. The use of killed cells has been restricted due to their limited stability. There is persistent demand to stabilize dead cells whilst retaining their immunological and structural properties. Though several efforts to preserve live cells are well documented, to our knowledge there are no reports available on dead cell preservation with retention of their immunological properties for immunization purpose.
Whole cells when used as vaccine generate better immune response when compared to cells lysates which is attributed to the repertoire of immunogenic epitopes, characterized or uncharacterized, on cell surface.
Hardev S. Pandha, Dorthe Cook, Rebecca Greenhalgh and Angus Dalgleish described use of killed cells (2005 BJU INTERNATIONAL, 95, 1336-1343) for Immunotherapy of murine prostate cancer. The immunogenicity of irradiated tumour cells is enhanced when they are killed ex-vivo using suicide-gene therapy.
Adjuvant is an agent that may stimulate the immune system and increase the response to a vaccine, without having any specific antigenic effect in itself (NCI, Definition). The word “adjuvant” comes from the Latin word adiuvare, meaning to help or aid. “An immunologic adjuvant is defined as any substance that acts to accelerate, prolong, or enhance antigen-specific immune responses when used in combination with specific vaccine antigens” (DNA Vaccines: Methods and Protocols, D. B. Lowrie and R. G. Whalen, Humana Press, 2000).
There are many known adjuvants in widespread use, including oils, aluminium salts, and virosomes, live or dead whole organisms, and extracts of microbes. The use of these adjuvants is also recommended in cancer vaccines to augment the immune stimulation by antigen/s.
U.S. Pat. No. 5,059,518 discloses the method of preservation of live Hybridoma cell lines, tissue cells and control cells for immunoassays and hematological measurements. The method comprises of isolation of Peripheral blood lymphocytes, resuspending cell pellet in phosphate buffered albumin and treating cells with isotonic solution of Intracellular cryopreservative Trehalose followed by lyophilisation. The lyophilates were resuspended in isotonic trehalose solution. Fluorescent Antibody labeled Cell Sorter (FACS) was used to characterize the antigenic determinants in Control and lyophilized cell population
U.S. Pat. No. 5,045,446 discloses preservation of live RBCs while retaining their metabolic activity. The patent discloses the use of intracellular cryopreservatives such as Galactose/Mannose/Xylose/Fructose/Glucose at concentration from (12.2-21.7%) along with extracellular PVP (of Mol. Wt. 10K-24K). The samples were rehydrated at 37° C. using 25.5% sucrose solution in Phosphate buffered Saline. Intact cell recovery was 52.9+7.9% after incorporating the polymer with carbohydrate Galactose, Mannose, Xylose, Fructose, Glucose, Trehalose and sucrose in lyophilisation solution showed marginal cell recovery. Carbohydrate (sucrose, trehalose, mannose, glucose in order of preference) was used at a concentration of 3.6% in reconstitution medium.
U.S. Pat. No. 5,648,206 reveals preservation of live RBCs with retained metabolic activity. The patent discloses the use of intracellular cryopreservative such as Galactose/Mannose/Xylose/Fructose/Glucose at concentration from (12.2-21.7%) with extracellular cryoprotactant PVP of Mol. Wt. 10K-24K. Lyophilisation medium contained monosaccharide (Xylose, glucose, mannose, ribose, fructose) at a concentration from 7%-37.5%. Extracellular cryoprotactant (PVP/Dextran) having a molecular weight from about 1K to 360K used at concentration of about 0.7%. The data of Trehalose combined with PVP is not disclosed though the patent claims that when trehalose and PVP were used together in lyophilzation solution they showed marginal cell recovery.
U.S. Pat. No. 5,425,951 discloses preservation of live RBCs retaining their metabolic activity. The patent describes the method of reconstituting lyophilized cells comprising the steps of treating cells with an aqueous solution containing a carbohydrate (Glucose/mannose; trehalose/sucrose) at a concentration of at least 1% and a polymer having a molecular weight of from IK to about 360K (PVP) at a concentration of about 20% by weight.
US Patent Application number 2005/0084481 A1 discloses preservation of mammalian and vertebrate cells, for example Macrophages and hematopoietic stem cells expressing P2X7 receptor.
European Patent EP0444159B1 discloses preservation of mammalian cells, hybridoma cell lines, tissue cells for immunoassays and other hematological measurements. Isotonic fluid containing 10% Trehalose was used for preserving proteinaceous markers on surface of mammalian cells. The steps involved resuspending the mammalian cells in phosphate buffered albumin and incubating the resultant pellet obtained after centrifugation in isotonic solution of 10% Trehalose at ambient temperature for approx. 30 min. This was followed by slow chilling at about −70° C. for approx. an hour.
During the slow freezing process, the extra cellular water will freeze first and salts will be left out, which will damage the cell membrane of dead cells. In case of live cells (eg. During cell culture) slow freezing is recommended (Biochimica et Biophysica Acta (BBA)—Biomembranes. Volume 1768, Issue 3, March 2007, Pages 728-736)
PCT publication WO97/04801 (PCT/US96/12251) discloses the use of sucrose and trehalose as cryo-preservatives. The invention encompasses anti-HER2 antibody. Histidine/Succinate buffer based pre-lyophilized formulation was developed to maintain pH of formulation. Polysorbate was added in pre-lyophilized formulation to reduce aggregation of reconstituted protein and formation of particulates. The patent also discloses the use of aromatic alcohols such as benzyl or phenol alcohol in reconstituting diluents. Trehalose based lyopreservative buffer was found to aid in stabilization of protein for 2 weeks at 40° C. and increase in trehalose concentration increased the stability for 1 year at 30° C. Addition of trehalose and sucrose also prevented aggregation at above said storage condition.
U.S. Pat. No. 5,759,774 discloses method of detecting circulating antibody types using dried or lyophilized cells. The patent discloses the preservation of mammalian cells notably RBCs, lymphocytes, platelets lipoosomes and hemosomes. Inventors used carbohydrate-polymer solution as lyophilisation preservative wherein the carbohydrate could be xylose mannose, glucose, ribose, mannose or fructose and Polymer could be PVP, HES or Dextran. The concentrations of monosaccharide pentoses and hexoses ranged from 7-37.5%. They also modified lyophilisation buffer composition through addition of glutathione inosine, adenine nicotinic acid glutamine, MgCl2.6H2O, Dextrose, PVP and HES and reconstitution buffer constituents by adding ATP, KH2PO4, Na2HPO4 and PVP.
European Patent EP 90906036 discloses method of lyophilisation of mammalian peripheral blood cells, cultured cells, hybridoma cell lines or tissue cells. The steps comprised of incubating the cell pellet, after centrifugation, in isotonic Trehalose solution followed by subjecting the cell suspension to freezing at −70° C. before lyophilisation. The lyophilized cells were reconstituted in distilled water.
PCT publication WO92/14359 (PCT/US92/00782) describes the method of lyophilisation of mammalian sperm cells. The patent discloses use of monosaccharide, preferably glucose in the concentration of about 0.1-2.6 M and polymer (or a mixture of polymers) with molecular weight preferably in the range of 1K to 350k. The preferred polymer was PVP followed by Dextran, HES and Poloxamers. PBS was used as lyophilisation buffer with pH in range of about 7.0 to 7.4. Suggested reconstituted medium comprised of Polymer (MW 15K) and PBS containing glucose and adenine. Typical cell metabolites such as ATP and NAD were incorporated along with monosaccharide such as xylose, glucose, ribose, mannose and fructose (at a concentration of 1M) in addition to glucose.
US Patent application 20080057040 discloses cryopreservation of stem cells. U.S. Pat. No. 5,071,741 discloses use of Agarose and alginate as non permeating and Glycerol and DMSO (at concentration of 1M) as permeating, in cryopreservation of cellular matter (bovine aortic endothelium derived cell line-BFA-Clone 1 & Islets of Langerhans from Murine Pancreas).
U.S. Pat. No. 4,004,975 discloses the Method of Isolating and Cryopreserving, at −80° C., Human White Cells from Whole Blood. The patent suggests combination of intracellular cryopreservative (5% DMSO) & extracellular cryopreservative (4% HES).
PCT publication WO 92/14360 (PCTUS92/00650) describes the method of lyophilisation and reconstitution of mixture of nucleated Non-Mammalian cells and blood matter. The method was invented with the aim of developing vaccines against Anaplasrna Marginal (Toxoplasma), The process streamlines continuous supply of blood samples infected with Analplasma spp (centrale) as the sample lyophilates can be stored for extended period of time. The lyophilisation mixture comprised of Monosaccharide (hexose and pentose) with at least two biocompatible amphipathic polymers. The monosaccharide was selected from the group consisting of Xylose, Glucose, Ribose, Mannose and fructose. The reconstitution buffer contained polymer at final concentration 0.7%.
The live cell membrane tonicity is maintained and we need some membrane pore opening agents/ATP to open up the pores for internalization of intracellular cryopreservant to replace water of hydration for preservation or cryopreservation (US2005/0084481 A1).
During live cell preservation it is also desirable to provide some carbon/ATP source (eg. Adenine) to keep them metabolically active (Advances in Biopreservation by John G. Baust) conversely it is also desirable to maintain adequate moisture content to keep them minimally metabolically active so that they can be revived later (US Patent Application 20100297231).
In case of live cell preservation there are chances of surface protein variability as a consequence of response to changing microenvironment due to addition of cryopreservants. However, dead cells may respond minimally (or not at all) to changing microenvironment conditions, because of defective/impaired protein synthesis machinery (Annual Review of Biophysics and Bioengineering. Vol. 3: 341-363).
It is desirable to preserve the integrity and immunogenicity of killed cells. The preservation of morphology and intactness of killed cells is useful for various purposes including developing immune mechanism associated therapies (Infect Immun. 1978 July; 21(1): 348) and diagnostics (Diagnostic Microbiology and Infectious Disease Volume 62, Issue 2, October 2008, Pages 133-141).
The direct influence of the osmotic stress on the cell membrane is documented for dead cells with observation of endocytotic vesiculation; membrane fluidity changes and increased membrane phase transition temperature. This Membrane phase transition which is believed to initiate lipid phase separation and membrane fusion may directly affect the viability of dehydrated dead cells. Also due to co-existence of the different phases during a phase transition, membrane permeability increases and cells may become leaky cellular contents during re-hydration will lead to reduction in intact cells number.
The method known for the preservation of live cells cannot be extended to preserving killed cells as                1. Dead cells loose membrane tonicity/plasticity and thus may collapse during freeze drying.        2. Dead cells forms aggregates which can be partially attributed to the presence of extracellular DNA of damaged dead cells.        3. The membrane integrity of internal organelles of dead cells is compromised and thus it is really challenging to maintain internal cellular architecture. (Morphological Features of Cell Death. News in Physiological Sciences, (2004) Vol. 19, No. 3, 124-128.)        4. Dead cells have different endocytotic vesiculation: membrane fluidity changes and membrane phase transition temperatures.        5. Dead cells respond minimally (or not at all) to changing microenvironment conditions, because of defective/impaired protein synthesis machinery.Morphology of a cell as defined herein relates to the size, shape and structure of cell.        
The present invention relates to a method of preserving killed cells. The method involves lyophilisation followed by reconstitution wherein the morphology, integrity and immunogenicity of the cells are retained even on prolonged storage. Further, pharmaceutical compositions comprising the killed cell retaining immunogenicity are disclosed. Such compositions find applications as Vaccines. The composition optionally contains one or more adjuvant to enhance the immunogenicity of the killed cells.